Accurate and rapid estimation of fluence, reflectance, and absorbance in multilayered biological media has been essential in many biophotonics applications that aim to diagnose, cure, or model in vivo tissue. The radiative transfer equation (RTE) rigorously models light transfer in absorbing and scattering media. However, analytical solutions to the RTE are limited even in simple homogeneous or plane media. Monte Carlo simulation has been used extensively to solve the RTE. However, Monte Carlo simulation is computationally intensive and may not be practical for applications that demand real-time results. Instead, the diffusion approximation has been shown to provide accurate estimates of light transport in strongly scattering tissue. The diffusion approximation is a greatly simplified model and produces analytical solutions for the reflectance and absorbance in tissue. However, the diffusion approximation breaks down if tissue is strongly absorbing, which is common in the visible part of the spectrum or in applications that involve darkly pigmented skin and/or high local volumes of blood such as port-wine stain therapy or reconstructive flap monitoring. In these cases, a model of light transfer that can accommodate both strongly and weakly absorbing regimes is required. Here we present a model of light transfer through layered biological media that represents skin with two strongly scattering and one strongly absorbing layer.

Monitoring of tissue blood volume and oxygen saturation using biomedical optics techniques has the potential to inform the assessment of tissue health, healing, and dysfunction. These quantities are typically estimated from the contribution of oxyhemoglobin and deoxyhemoglobin to the absorption spectrum of the dermis. However, estimation of blood related absorption in superficial tissue such as the skin can be confounded by the strong absorption of melanin in the epidermis. Furthermore, epidermal thickness and pigmentation varies with anatomic location, race, gender, and degree of disease progression. This study describes a technique for decoupling the effect of melanin absorption in the epidermis from blood absorption in the dermis for a large range of skin types and thicknesses. An artificial neural network was used to map input optical properties to spatial frequency domain diffuse reflectance of two layer media. Then, iterative fitting was used to determine the optical properties from simulated spatial frequency domain diffuse reflectance. Additionally, an artificial neural network was trained to directly map spatial frequency domain reflectance to sets of optical properties of a two layer medium, thus bypassing the need for iteration. In both cases, the optical thickness of the epidermis and absorption and reduced scattering coefficients of the dermis were determined independently. The accuracy and efficiency of the iterative fitting approach was compared with the direct neural network inversion.

Background and objective

This paper compares the ability of three analytic techniques to predict chromophore concentration from fluorescence emission spectra of homogeneous, turbid samples with optical properties similar to human tissue.

Study design, materials and methods

Two models of light propagation were implemented (exponential attenuation, two flux Kubelka-Munk theory); a priori information about sample optical properties was used to analyze data with the two flux Kubelka-Munk model. The third data analysis technique utilizes the method of partial least squares (PLS) to develop an empirical, linear model of sample fluorescence from a training set with optical properties and known concentrations representative of those to be predicted. This model can be applied to predict chromophore concentrations in the unknown samples.

Results

Of the three methods, PLS achieved the most accurate results and was able to predict fluorophore concentration to within +/- 6% of true values.

Conclusion

We investigated conditions under which PLS predictions were most accurate and find that best results are achieved when predictions are based on fluorescence emission spectra at more than one excitation wavelength with inclusion of the tail of Rayleigh scattering at the excitation wavelength.

We develop a superficial diffusing probe with a 3 mm source-detector separation that can be used in combination with diffuse optical spectroscopic (DOS) methods to noninvasively determine full-spectrum optical properties of superficial in vivo skin in the wavelength range from 650 to 1000 nm. This new probe uses a highly scattering layer to diffuse photons emitted from a collimated light source and relies on a two-layer diffusion model to determine tissue absorption coefficient mu a and reduced scattering coefficient mu's. By employing the probe to measure two-layer phantoms that mimic the optical properties of skin, we demonstrate that the probe has an interrogation depth of 1 to 2 mm. We carry out SSFDPM (steady state frequency-domain photon migration) measurements using this new probe on the volar forearm and palm of 15 subjects, including five subjects of African descent, five Asians, and five Caucasians. The optical properties of in vivo skin determined using the superficial diffusing probe show considerable similarity to published optical properties of carefully prepared ex vivo epidermis+dermis.

A fiber-optic probe-based instrument, designed for assessment of parameters related to microcirculation, red blood cell tissue fraction (fRBC), oxygen saturation (SO2), and speed resolved perfusion, has been evaluated using state-of-the-art tissue phantoms. The probe integrates diffuse reflectance spectroscopy (DRS) at two source-detector separations and laser Doppler flowmetry, using an inverse Monte Carlo method for identifying the parameters of a multilayered tissue model. Here, we characterize the accuracy of the DRS aspect of the instrument using (1) liquid blood phantoms containing yeast and (2) epidermis-dermis mimicking solid-layered phantoms fabricated from polydimethylsiloxane, titanium oxide, hemoglobin, and coffee. The root-mean-square (RMS) deviations for fRBC for the two liquid phantoms were 11% and 5.3%, respectively, and 11% for the solid phantoms with highest hemoglobin signatures. The RMS deviation for SO2 was 5.2% and 2.9%, respectively, for the liquid phantoms, and 2.9% for the solid phantoms. RMS deviation for the reduced scattering coefficient (μs'), for the solid phantoms was 15% (475 to 850 nm). For the liquid phantoms, the RMS deviation in average vessel diameter (D) was 1  μm. In conclusion, the skin microcirculation parameters fRBC and SO2, as well as, μs' and D are estimated with reasonable accuracy.

We present a novel, noncontact method for the determination of quantitative optical properties of turbid media from 430 to 1050 nm. Through measuring the broadband reflectance from an unknown sample as a function of the spatial frequency of the projected illumination patterns, the absolute absorption and reduced scattering coefficients can be calculated without a priori assumptions of the chromophores present. This technique, which is called spatially modulated quantitative spectroscopy (SMoQS), was validated through the quantification of optical properties of homogenous liquid phantoms with known concentrations of absorbers and scatterers. The properties of the phantoms were recovered across the range of values prepared with R(2) values of 0.985 and 0.996 for absorption and reduced scattering, respectively. A measurement was also performed on skin tissue as a demonstration of the method's performance in vivo. The resultant absorption spectrum was well described by a multichromophore fit, and the quantitative values for oxy- and deoxyhemoglobin, water, and melanin were within published ranges for skin.

We design a special diffusing probe to investigate the optical properties of human skin in vivo. The special geometry of the probe enables a modified two-layer (MTL) diffusion model to precisely describe the photon transport even when the source-detector separation is shorter than 3 mean free paths. We provide a frequency domain comparison between the Monte Carlo model and the diffusion model in both the MTL geometry and conventional semiinfinite geometry. We show that using the Monte Carlo model as a benchmark method, the MTL diffusion theory performs better than the diffusion theory in the semiinfinite geometry. In addition, we carry out Monte Carlo simulations with the goal of investigating the dependence of the interrogation depth of this probe on several parameters including source-detector separation, sample optical properties, and properties of the diffusing high-scattering layer. From the simulations, we find that the optical properties of samples modulate the interrogation volume greatly, and the source-detector separation and the thickness of the diffusing layer are the two dominant probe parameters that impact the interrogation volume. Our simulation results provide design guidelines for a MTL geometry probe.

We have developed a method for extracting spatial frequency information content from biological tissue, which is used to calculate tissue optical properties and determine tissue structural orientation. This demodulation method employs a two-dimensional Hilbert transform using a spiral phase function in Fourier space. The approach presented here allows for the determination of tissue optical properties using a single frame of data for each modulation frequency, increasing imaging speed by two to threefold versus conventional, three-phase spatial frequency domain imaging (SFDI). This new single-phase Hilbert transform approach recovers optical property and scattering orientation index values within 1% and 10% of three-phase SFDI, respectively. These results suggest that, using the Hilbert demodulation technique, SFDI data acquisition speed can be increased significantly while preserving data quality, which will help us move forward toward the implementation of a real-time SFDI platform.

Monitoring of tissue blood volume and local oxygen saturation can inform the assessment of tissue health, healing, and dysfunction. These quantities can be estimated from the contribution of oxyhemoglobin and deoxyhemoglobin to the absorption spectrum of the dermis. However, estimation of blood related absorption in skin can be confounded by the strong absorption of melanin in the epidermis and epidermal thickness and pigmentation varies with anatomic location, race, gender, and degree of disease progression. Therefore, a method is desired that decouples the effect of melanin absorption in the epidermis from blood absorption in the dermis for a large range of skin types and thicknesses. A previously developed inverse method based on a neural network forward model was applied to simulated spatial frequency domain reflectance of skin for multiple wavelengths in the near infrared. It is demonstrated that the optical thickness of the epidermis and absorption and reduced scattering coefficients of the dermis can be determined independently and with minimal coupling. Then, the same inverse method was applied to reflectance measurements from a tissue simulating phantom and in vivo human skin. Oxygen saturation and total hemoglobin concentrations were estimated from the volar forearms of weakly and strongly pigmented subjects using a standard homogeneous model and the present two layer model.

We have developed a method for extracting spatial frequency information content from biological tissue, which is used to calculate tissue optical properties and determine tissue structural orientation. This demodulation method employs a two-dimensional Hilbert transform using a spiral phase function in Fourier space. The approach presented here allows for the determination of tissue optical properties using a single frame of data for each modulation frequency, increasing imaging speed by two to threefold versus conventional, three-phase spatial frequency domain imaging (SFDI). This new single-phase Hilbert transform approach recovers optical property and scattering orientation index values within 1% and 10% of three-phase SFDI, respectively. These results suggest that, using the Hilbert demodulation technique, SFDI data acquisition speed can be increased significantly while preserving data quality, which will help us move forward toward the implementation of a real-time SFDI platform.